INO1 Expression Research Articles

The Cellular Functions of the Yeast Lipin Homolog Pah1p Are Dependent on Its Phosphatidate Phosphatase Activity

The Saccharomyces cerevisiae PAH1-encoded Mg2+-dependent phosphatidate phosphatase (PAP1, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol and Pi. This enzyme plays a major role in the synthesis of triacylglycerols and phospholipids in S. cerevisiae. PAP1 contains the DXDX(T/V) catalytic motif (DIDGT at residues 398-402) that is shared by the mammalian fat-regulating protein lipin 1 and the superfamily of haloacid dehalogenase-like proteins. The yeast enzyme also contains a conserved glycine residue (Gly80) that is essential for the fat-regulating function of lipin 1 in a mouse model. In this study, we examined the roles of the putative catalytic motif and the conserved glycine for PAP1 activity by a mutational analysis. The PAP1 activities of the D398E and D400E mutant enzymes were reduced by >99.9%, and the activity of the G80R mutant enzyme was reduced by 98%. The mutant PAH1 alleles whose products lacked PAP1 activity were nonfunctional in vivo and failed to complement the pah1Delta mutant phenotypes of temperature sensitivity, respiratory deficiency, nuclear/endoplasmic reticulum membrane expansion, derepression of INO1 expression, and alterations in lipid composition. These results demonstrated that the PAP1 activity of the PAH1 gene product is essential for its roles in lipid metabolism and cell physiology.

Transcriptional regulation of phospholipid biosynthesis is linked to fatty acid metabolism by an acyl-CoA-binding-protein-dependent mechanism in Saccharomyces cerevisiae

In the present study, we have used DNA microarray and quantitative real-time PCR analysis to examine the transcriptional changes that occur in response to cellular depletion of the yeast acyl-CoA-binding protein, Acb1p. Depletion of Acb1p resulted in the differential expression of genes encoding proteins involved in fatty acid and phospholipid synthesis (e.g. FAS1, FAS2, ACC1, OLE1, INO1 and OPI3), glycolysis and glycerol metabolism (e.g. GPD1 and TDH1), ion transport and uptake (e.g. ITR1 and HNM1) and stress response (e.g. HSP12, DDR2 and CTT1). In the present study, we show that transcription of the INO1 gene, which encodes inositol-3-phosphate synthase, cannot be fully repressed by inositol and choline, and UAS(INO1) (inositol-sensitive upstream activating sequence)-driven transcription is enhanced in Acb1p-depleted cells. In addition, the reduction in inositol-mediated repression of INO1 transcription observed after depletion of Acb1p appeared to be independent of the transcriptional repressor, Opi1p. We also demonstrated that INO1 and OPI3 expression can be normalized in Acb1p-depleted cells by the addition of high concentrations of exogenous fatty acids, or by the overexpression of FAS1 or ACC1. Together, these findings revealed an Acb1p-dependent connection between fatty acid metabolism and transcriptional regulation of phospholipid biosynthesis in yeast. Finally, expression of an Acb1p mutant which is unable to bind acyl-CoA esters could not normalize the transcriptional changes caused by Acb1p depletion. This strongly implied that gene expression is modulated either by the Acb1p-acyl-CoA ester complex directly or by its ability to donate acyl-CoA esters to utilizing systems.

Disruption of aldo-keto reductase genes leads to elevated markers of oxidative stress and inositol auxotrophy in Saccharomyces cerevisiae

A large family of aldo-keto reductases with similar kinetic and structural properties but unknown physiological roles is expressed in the yeast Saccharomyces cerevisiae. Strains with one or two AKR genes disrupted have apparently normal phenotypes, but disruption of at least three AKR genes results in a heat shock phenotype and slow growth in inositol-deficient culture medium (Ino−). The present study was carried out to identify metabolic or signaling defects that may underlie phenotypes that emerge in AKR deficient strains. Here we demonstrate that pretreatment of a pentuple AKR null mutant with the anti-oxidative agent N-acetyl-cysteine rescues the heat shock phenotype. This indicates that AKR gene disruption may be associated with defects in oxidative stress response. We observed additional markers of oxidative stress in AKR-deficient strains, including reduced glutathione levels, constitutive nuclear localization of the oxidation-sensitive transcription factor Yap1 and upregulation of a set of Yap1 target genes whose function as a group is primarily involved in response to oxidative stress and redox balance. Genetic analysis of the Ino− phenotype of the null mutants showed that defects in transcriptional regulation of the INO1, which encodes for inositol-1-phosphate synthase, can be rescued through ectopic expression of a functional INO1. Taken together, these results suggest potential roles for AKRs in oxidative defense and transcriptional regulation.

Glycogen synthase kinase‐3 is required for optimal de novo synthesis of inositol

Studies have shown that the inositol biosynthetic pathway and the enzyme glycogen synthase kinase-3 (GSK-3) are targets of the mood-stabilizing drugs lithium and valproate. However, a relationship between these targets has not been previously described. We hypothesized that GSK-3 may play a role in inositol synthesis, and that loss of GSK-3 may lead to inositol depletion, thus providing a mechanistic link between the two drug targets. Utilizing a yeast Saccharomyces cerevisiae gsk-3Delta quadruple-null mutant, in which all four genes encoding homologues of mammalian GSK-3 are disrupted, we tested the hypothesis that GSK-3 is required for de novo inositol biosynthesis. The gsk-3Delta mutant exhibited multiple features of inositol depletion, including defective growth in inositol-lacking medium, decreased intracellular inositol, increased INO1 and ITR1 expression, and decreased levels of phosphatidylinositol. Treatment of wild-type cells with a highly specific GSK-3 inhibitor led to a significant increase in INO1 expression. Supplementation with inositol alleviated the temperature sensitivity of gsk-3Delta. Activity of myo-inositol-3 phosphate synthase, the rate-limiting enzyme in inositol de novo biosynthesis, was decreased in gsk-3Delta. These results demonstrate for the first time that GSK-3 is required for optimal myo-inositol-3 phosphate synthase activity and de novo inositol biosynthesis, and that loss of GSK-3 activity causes inositol depletion.

Enhanced levels of Pis1p (phosphatidylinositol synthase) improve the growth of Saccharomyces cerevisiae cells deficient in Rsp5 ubiquitin ligase

The Rsp5 ubiquitin ligase plays a role in many cellular processes including the biosynthesis of unsaturated fatty acids. The PIS1 (phosphatidylinositol synthase gene) encoding the enzyme Pis1p which catalyses the synthesis of phosphatidylinositol from CDP-diacyglycerol and inositol, was isolated in a screen for multicopy suppressors of the rsp5 temperature sensitivity phenotype. Suppression was allele non-specific. Interestingly, expression of PIS1 was 2-fold higher in the rsp5 mutant than in wild-type yeast, whereas the introduction of PIS1 in a multicopy plasmid increased the level of Pis1p 6-fold in both backgrounds. We demonstrate concomitantly that the expression of INO1 (inositol phosphate synthase gene) was also elevated approx. 2-fold in the rsp5 mutant as compared with the wild-type, and that inositol added to the medium improved growth of rsp5 mutants at a restrictive temperature. These results suggest that enhanced phosphatidylinositol synthesis may account for PIS1 suppression of rsp5 defects. Analysis of lipid extracts revealed the accumulation of saturated fatty acids in the rsp5 mutant, as a consequence of the prevention of unsaturated fatty acid synthesis. Overexpression of PIS1 did not correct the cellular fatty acid content; however, saturated fatty acids (C(16:0)) accumulated preferentially in phosphatidylinositol, and (wild-type)-like fatty acid composition in phosphatidylethanolamine was restored.

Role of signal transduction pathways in the regulation of lipid metabolism in S. cerevisiae

The INO1 gene is the most highly regulated of a group of co-regulated genes involved in phospholipid biosynthesis in yeast that contain the repeated element UASINO in their promoter. Our observations suggest that the complex regulation of INO1 and other UASINO-containing genes is due to the combinatorial inputs of several signal transduction pathways. We report here that mutants defective in the PKC-mediated cell integrity pathway, including pkc1Δ, bck1Δ and mpk1Δ, exhibit inositol auxotrophy (Ino−phenotype), indicative of defects in INO1 expression. The opi1 mutation confers high-level constitutive INO1 expression, indicated by the Opi− phenotype (for overproduction of inositol). Deletion of the OPI1 gene in the pck1, bck1, and mpk1 mutants suppresses their Ino− phenotype, and confers an Opi−phenotype. Our preliminary results indicate that the mpk1 mutation results in abnormal INO1 expression. Deleting MPK1 also results in an abnormal accumulation of certain lipid species. A screen for high copy suppressors of the mpk1Δ Ino− phenotype identified NTE1. The NTE1 gene encodes a phospholipase type B that deacylates phosphatidylcholine (PC) to glycerophosphocholine. The potential PKC pathway-mediated regulation of lipid turnover, specifically deacylation of PC, is currently under investigation. Supported by a grant of the National Institutes of Health to S.A.H. (GM019629-31).

Role of the unfolded protein response pathway in secretory stress and regulation of INO1 expression in Saccharomyces cerevisiae.

The unfolded protein response pathway (UPR) enables the cell to cope with the buildup of unfolded proteins in the endoplasmic reticulum (ER). UPR loss-of-function mutants, hac1Delta and ire1Delta, are also inositol auxotrophs, a phenotype associated with defects in expression of INO1, the most highly regulated of a set of genes encoding enzymes of phospholipid metabolism. We now demonstrate that the UPR plays a functional role in membrane trafficking under conditions of secretory stress in yeast. Mutations conferring a wide range of membrane trafficking defects exhibited negative genetic interaction when combined with ire1Delta and hac1Delta. At semipermissive temperatures, carboxypeptidase Y transit time to the vacuole was slower in Sec(-) cells containing an ire1Delta or hac1Delta mutation than in Sec(-) cells with an intact UPR. The UPR was induced in Sec(-) cells defective in subcellular membrane trafficking events ranging from ER vesicle trafficking to distal secretion and in erg6Delta cells challenged with brefeldin A. However, the high levels of UPR induction observed under these conditions were not correlated with elevated INO1 expression. Indeed, many of the Sec(-) mutants that had elevated UPR expression at semipermissive growth temperatures failed to achieve wild-type levels of INO1 expression under these same conditions.

Valproate disrupts regulation of inositol responsive genes and alters regulation of phospholipid biosynthesis.

Valproate (VPA) is one of the two drugs approved by the Food and Drug Administration (FDA) for the treatment of bipolar disorder. The therapeutic mechanism of VPA has not been established. We have shown previously that growth of the yeast Saccharomyces cerevisiae in the presence of VPA causes a decrease in intracellular inositol and inositol-1-P, and a dramatic increase in expression of INO1, which encodes the rate limiting enzyme for de novo inositol biosynthesis. To understand the underlying mechanism of action of VPA, INO1, CHO1 and INO2 expression, intracellular inositol and phospholipid biosynthesis were studied as a function of acute and chronic exposure of growing cells to the drug. A decrease in intracellular inositol was apparent immediately after addition of VPA. Surprisingly, expression of genes that are usually derepressed during inositol depletion, including INO1, CHO1 and INO2 (that contain inositol-responsive UASINO sequences) decreased several fold during the first hour, after which expression began to increase. Incorporation of 32Pi into total phospholipids was significantly decreased. Pulse labelling of CDP-DG and PG, shown previously to increase during inositol depletion, increased within 30 min. However, pulse labelling of PS, which normally increases during inositol depletion, was decreased within 30 min. PS synthase activity in cell extracts decreased with time, although VPA did not directly inhibit PS synthase enzyme activity. Thus, in contrast to the effect of chronic VPA treatment, short-term exposure to VPA abrogated the normal response to inositol depletion of inositol responsive genes and led to aberrant synthesis of phospholipids.

Phosphorylation of the Yeast Phospholipid Synthesis Regulatory Protein Opi1p by Protein Kinase A

The Opi1p transcription factor plays a negative regulatory role in the expression of UASINO-containing genes involved in phospholipid synthesis in the yeast Saccharomyces cerevisiae. The phosphorylation of Opi1p by protein kinase A (cAMP-dependent protein kinase) was examined in this work. Using a maltose-binding protein-Opi1p fusion protein as a substrate, protein kinase A activity was time- and dose-dependent and dependent on the concentrations of Opi1p and ATP. Protein kinase A phosphorylated Opi1p on multiple serine residues. The synthetic peptides SCRQKSQPSE and SQVRESLLNL containing the protein kinase A motif for Ser31 and Ser251, respectively, within Opi1p were substrates for protein kinase A. Phosphorylation of S31A and S251A mutant maltose-binding protein-Opi1p fusion proteins by protein kinase A was reduced when compared with the wild type protein, and phosphopeptides present in wild type Opi1p were absent from the S31A and S251A mutant proteins. In vivo labeling experiments showed that the extent of phosphorylation of the S31A and S251A mutant proteins was reduced when compared with the wild type protein. The physiological consequence of the phosphorylation of Opi1p at Ser31 and Ser251 was examined by measuring the effects of the S31A and S251A mutations on the expression of the UASINO-containing gene INO1. The beta-galactosidase activity driven by an INO1-CYC-lacZ reporter gene in opi1Delta mutant cells expressing the S31A and S251A mutant Opi1p proteins was elevated 42 and 35%, respectively, in the absence of inositol and 55 and 52%, respectively, in the presence of inositol when compared with cells expressing wild type Opi1p. These data supported the conclusion that phosphorylation of Opi1p at Ser31 and Ser251 mediated the stimulation of the negative regulatory function of Opi1p on the expression of the INO1 gene.

The effect of lithium on expression of genes for inositol biosynthetic enzymes in mouse hippocampus; a comparison with the yeast model

In the de novo synthesis of inositol, the conversion of d-glucose-6-phosphate to l- myo-inositol-1-phosphate (MIP) is catalyzed by MIP synthase. Little is known about mammalian MIP synthase and nothing is known about its regulation. The second step in inositol biosynthesis is the conversion of MIP to inositol by inositol-monophosphatase (IMPase), a common step to inositol production via the de novo pathway and its recycling from inositol phosphates. Because lithium inhibits IMPase both in yeast and in mammals, and the drug upregulates yeast MIP synthase ( INO1) and downregulates IMPase ( INM1), the present study was undertaken to determine whether chronic in vivo therapeutic lithium concentrations affect MIP synthase and IMPase expression in mouse frontal cortex and hippocampus. Mice were treated with food containing LiCl (2.5 g/kg) for 10 days. RNA was purified from the brain areas and mRNA amplified using RT-PCR. Expression of MIP synthase and IMPA1 (one of the genes coding for IMPase) but not IMPA2 was upregulated in mouse hippocampus. None of the genes were affected in the frontal cortex. In yeast, when inositol is limiting, the heterodimeric transcriptional activator Ino2p/Ino4p derepresses expression of INO1 by binding to the upstream activation sequence UAS INO. Using the TFSEARCH program, we found that the promoter of the virtual human MIP synthase gene contains upstream stimulating factor (USF) elements with a similar core binding sequence. The fact that lithium treatment upregulates both MIP synthase and IMPA1 mRNA levels in mouse hippocampus may reflect a compensatory response of both genes to inositol depletion.

Role of the unfolded protein response pathway in regulation of INO1 and in the sec14 bypass mechanism in Saccharomyces cerevisiae.

INO1, encoding inositol 1-phosphate synthase, is the most highly regulated of a class of genes containing the repeated element, UAS(INO), in their promoters. Transcription of UAS(INO)-containing genes is modulated by the availability of exogenous inositol and by signals generated by alteration of phospholipid metabolism. The unfolded protein response (UPR) pathway also is involved in INO1 expression and the ire1Delta and hac1Delta mutants are inositol auxotrophs. We examined the role of the UPR in transmitting a signal generated in response to inositol deprivation and to alteration of phospholipid biosynthesis created in the sec14(ts) cki1Delta genetic background. We report that the UPR is required for sustained high-level INO1 expression in wild-type strains, but not for transient derepression in response to inositol deprivation. Moreover, the UPR is not required for expression or regulation of INO1 in response to the change in lipid metabolism that occurs in the sec14(ts) cki1Delta genetic background. Thus, the UPR signal transduction pathway is not involved directly in transcriptional regulation of INO1 and other UAS(INO)-containing genes. However, we discovered that inactivation of Sec14p leads to activation of the UPR, and that sec14 cki1 strains exhibit defective vacuolar morphology, suggesting that the mechanism by which the cki1Delta mutation suppresses the growth and secretory defect of sec14 does not fully restore wild-type morphology. Finally, synthetic lethality involving sec14 and UPR mutations suggests that the UPR plays an essential role in survival of sec14 cki1 strains.

Epi-inositol regulates expression of the yeast INO1 gene encoding inositol-1-P synthase

Myo-inositol exerts behavioral effects in animal models of psychiatric disorders and is effective in clinical trials in psychiatric patients. Interestingly, epi-inositol exerts behavioral effects similar to myo-inositol, even though epi-inositol is not a substrate for synthesis of phosphatidylinositol. We postulated that the behavioral effects of epi-inositol may be due to its effects on gene expression. Yeast INO1expression was measured in northern blots. INM1 was determined by beta-galactosidase activity in a strain containing the fusion gene INM1-lacZintegrated into the genome. Epi-inositol affects regulation of expression of the INO1 gene (encoding inositol-1-P synthase), even though it cannot support growth of an inositol auxotroph (suggesting that, as in mammalian cells, it is not incorporated into phosphatidylinositol). Like myo-inositol, although to a lesser extent, epi-inositol causes a significant reduction in INO1 expression, and reverses the lithium- or valproate-induced increase in INO1 expression. However, it does not affect regulation of INM1 (encoding inositol monophosphatase), the expression of which is up-regulated by myo-inositol. The observed regulatory effects of epi-inositol on expression of the most highly regulated gene in the inositol biosynthetic pathway may help to explain how this inositol isomer can exert behavioral effects without being incorporated into phosphatidylinositol.

Inhibition of acetyl coenzyme A carboxylase activity restores expression of the INO1 gene in a snf1 mutant strain of Saccharomyces cerevisiae.

Mutations in the Saccharomyces cerevisiae SNF1 gene affect a number of cellular processes, including the expression of genes involved in carbon source utilization and phospholipid biosynthesis. To identify targets of the Snf1 kinase that modulate expression of INO1, a gene required for an early, rate-limiting step in phospholipid biosynthesis, we performed a genetic selection for suppressors of the inositol auxotrophy of snf1Delta strains. We identified mutations in ACC1 and FAS1, two genes important for fatty acid biosynthesis in yeast; ACC1 encodes acetyl coenzyme A carboxylase (Acc1), and FAS1 encodes the beta subunit of fatty acid synthase. Acc1 was shown previously to be phosphorylated and inactivated by Snf1. Here we show that snf1Delta strains with increased Acc1 activity exhibit decreased INO1 transcription. Strains carrying the ACC1 suppressor mutation have reduced Acc1 activity in vitro and in vivo, as revealed by enzymatic assays and increased sensitivity to the Acc1-specific inhibitor soraphen A. Moreover, a reduction in Acc1 activity, caused by addition of soraphen A, provision of exogenous fatty acid, or conditional expression of ACC1, suppresses the inositol auxotrophy of snf1Delta strains. Together, these findings indicate that the inositol auxotrophy of snf1Delta strains arises in part from elevated Acc1 activity and that a reduction in this activity restores INO1 expression in these strains. These results reveal a Snf1-dependent connection between fatty acid production and phospholipid biosynthesis, identify Acc1 as a Snf1 target important for INO1 transcription, and suggest models in which metabolites that are generated or utilized during fatty acid biosynthesis can significantly influence gene expression in yeast.

Phosphorylation of the Yeast Phospholipid Synthesis Regulatory Protein Opi1p by Protein Kinase C

Opi1p is a negative regulator of expression of phospholipid-synthesizing enzymes in the yeast Saccharomyces cerevisiae. In this work, we examined the phosphorylation of Opi1p by protein kinase C. Using a purified maltose-binding protein-Opi1p fusion protein as a substrate, protein kinase C activity was time- and dose-dependent, and dependent on the concentrations of Opi1p and ATP. Protein kinase C phosphorylated Opi1p on a serine residue. The Opi1p synthetic peptide GVLKQSCRQK, which contained a protein kinase C sequence motif at Ser(26), was a substrate for protein kinase C. Phosphorylation of a purified S26A mutant maltose-binding protein-Opi1p fusion protein by the kinase was reduced when compared with the wild-type protein. A major phosphopeptide present in purified wild-type Opi1p was absent from the purified S26A mutant protein. In vivo labeling experiments showed that the phosphorylation of Opi1p was physiologically relevant, and that the extent of phosphorylation of the S26A mutant protein was reduced by 50% when compared with the wild-type protein. The physiological consequence of the phosphorylation of Opi1p at Ser(26) was examined by measuring the effect of the S26A mutation on the expression of the phospholipid synthesis gene INO1. The beta-galactosidase activity driven by an INO1-CYC-lacI'Z reporter gene in opi1Delta mutant cells expressing the S26A mutant Opi1p was about 50% lower than that of cells expressing the wild-type Opi1p protein. These data supported the conclusion that phosphorylation of Opi1p at Ser(26) mediated the attenuation of the negative regulatory function of Opi1p on the expression of the INO1 gene.

Over-expression of the INO2 regulatory gene alters regulation of an INO1-lacZ reporter gene but does not affect regulation of INO1 expression

The yeast INO2 gene encodes a transcriptional activator. Inositol and choline repress transcription of the INO2 gene, and its target genes. That is, INO2 transcription is auto-regulated. This observation prompted two separate investigations to determine if regulation of INO2 is required for regulation of its target genes. One study, using northern blot hybridization, showed that constitutive INO2 transcription did not affect regulation of the INO1 gene, while another study revealed that it severely dampened regulation of an INO1-lacZ gene. By repeating both assays from a single yeast strain we demonstrate that this disparity is due to the different reporter systems.

Lithium and Valproate Decrease Inositol Mass and Increase Expression of the Yeast INO1 and INO2Genes for Inositol Biosynthesis

Bipolar affective disorder (manic-depressive illness) is a chronic, severe, debilitating illness affecting 1-2% of the population. The Food and Drug Administration-approved drugs lithium and valproate are not completely effective in the treatment of this disorder, and the mechanisms underlying their therapeutic effects have not been established. We are employing genetic and molecular approaches to identify common targets of lithium and valproate in the yeast Saccharomyces cerevisiae. We show that both drugs affect molecular targets in the inositol metabolic pathway. Lithium and valproate cause a decrease in intracellular myo-inositol mass and an increase in expression of both a structural (INO1) and a regulatory (INO2) gene required for inositol biosynthesis. The opi1 mutant, which exhibits constitutive expression of INO1, is more resistant to inhibition of growth by lithium but not by valproate, suggesting that valproate may inhibit the Ino1p-catalyzed synthesis of inositol 1-phosphate. Consistent with this possibility, growth in valproate leads to decreased synthesis of inositol monophosphate. Thus, both lithium and valproate perturb regulation of the inositol biosynthetic pathway, albeit via different mechanisms. This is the first demonstration of increased expression of genes in the inositol biosynthetic pathway by both lithium and valproate. Because inositol is a key regulator of many cellular processes, the effects of lithium and valproate on inositol synthesis have far-reaching implications for predicting genetic determinants of responsiveness and resistance to these agents.

Regulation of the yeast INO1 gene. The products of the INO2, INO4 and OPI1 regulatory genes are not required for repression in response to inositol.

The ino2Delta, ino4Delta, opi1Delta, and sin3Delta mutations all affect expression of INO1, a structural gene for inositol-1-phosphate synthase. These same mutations affect other genes of phospholipid biosynthesis that, like INO1, contain the repeated element UAS(INO) (consensus 5' CATGTGAAAT 3'). In this study, we evaluated the effects of these four mutations, singly and in all possible combinations, on growth and expression of INO1. All strains carrying an ino2Delta or ino4Delta mutation, or both, failed to grow in medium lacking inositol. However, when grown in liquid culture in medium containing limiting amounts of inositol, the opi1Delta ino4Delta strain exhibited a level of INO1 expression comparable to, or higher than, the wild-type strain growing under the same conditions. Furthermore, INO1 expression in the opi1Delta ino4Delta strain was repressed in cells grown in medium fully supplemented with both inositol and choline. Similar results were obtained using the opi1Delta ino2Delta ino4Delta strain. Regulation of INO1 was also observed in the absence of the SIN3 gene product. Therefore, while Opi1p, Sin3p, and the Ino2p/Ino4p complex all affect the overall level of INO1 expression in an antagonistic manner, they do not appear to be responsible for transmitting the signal that leads to repression of INO1 in response to inositol. Various models for Opi1p function were tested and no evidence for binding of Opi1p to UAS(INO), or to Ino2p or Ino4p, was obtained.

The REG1 gene product is required for repression of INO1 and other inositol-sensitive upstream activating sequence-containing genes of yeast.

A search was conducted for suppressors of the inositol auxotrophic phenotype of the ino4-8 mutant of yeast. The ino4-8 mutation is a single base pair change that results in substitution of lysine for glutamic acid at position 79 in the bHLH domain of the yeast regulatory protein, Ino4p. Ino4p dimerizes with a second bHLH protein, Ino2p, to form a complex that binds to the promoter of the INO1 gene, activating transcription. Of 31 recessive suppressors of ino4-8 isolated, 29 proved to be alleles of a single locus, identified as REG1, which encodes a regulatory subunit of a protein phosphatase involved in the glucose response pathway. The suppressor mutation, sia1-1, identified as an allele of REG1, caused constitutive INO1 expression and was capable of suppressing the inositol auxotrophy of a second ino4 missense mutant, ino4-26, as well as ino2-419, a missense mutation of INO2. The suppressors analyzed were unable to suppress ino2 and ino4 null mutations, but the reg1 deletion mutation could suppress ino4-8. A deletion mutation in the OPI1 negative regulator was incapable of suppressing ino4-8. The relative roles of the OPI1 and REG1 gene products in control of INO1 expression are discussed.

Functional characterization of the repeated UASINO element in the promoters of the INO1 and CHO2 genes of yeast

In yeast, INO1 and CHO2 gene expression is subject to repression in response to inositol and choline supplementation. The response by both genes to inositol is controlled by a single set of regulatory factors and the highly conserved and repeated UASINO element (consensus: 5' CATGTGAAAT 3') that is found in multiple copies in both promoters. However, none of the native elements found in the INO1 and CHO2 promoters constitutes an exact match to the consensus element and the functionality of individual elements from these two promoters has not been tested. In this study, the function of individual putative UASINO elements from both promoters was tested by placing promoter fragments into a reporter construct which lacked a UAS element but contained the TATA element and start of transcription from the yeast CYC1 gene fused to the Escherichia coli lacZ gene. In addition, a set of oligonucleotides containing the consensus UASINO element with the first position systematically modified was also tested for UASINO function. These studies indicated that elements that contain a C or an A as the first base at the 5' end are functional to varying degrees. The majority of potential UASINO elements from the INO1 promoter were found to be inactive, whereas all of the elements from the CHO2 promoter tested were active. These results are discussed in light of the differential regulation of the two promoters.

The Yeast BSD2-1 Mutation Influences Both the Requirement for Phosphatidylinositol Transfer Protein Function and Derepression of Phospholipid Biosynthetic Gene Expression in Yeast

The BSD2-1 allele renders Saccharomyces cerevisiae independent of its normally essential requirement for phosphatidylinositol transfer protein (Sec14p) in the stimulation of Golgi secretory function and cell viability. We now report that BSD2-1 yeast mutants also exhibit yet another phenotype, an inositol auxotrophy. We demonstrate that the basis for this Ino- phenotype is the inability of BSD2-1 strains to derepress transcription of INO1, the structural gene for the enzyme that catalyzes the committed step in de novo inositol biosynthesis in yeast. This constitutive repression of INO1 expression is mediated through specific inactivation of Ino2p, a factor required for trans-activation of INO1 transcription, and we show that these transcriptional regulatory defects can be uncoupled from the "bypass Sec14p" phenotype of BSD2-1 strains. Finally, we present evidence that newly synthesized phosphatidylinositol is subject to accelerated turnover in BSD2-1 mutants and that prevention of this accelerated phosphatidyl-inositol turnover in turn negates suppression of Sec14p defects by BSD2-1. We propose that, in BSD2-1 strains, a product(s) generated by phosphatidylinositol turnover coordinately modulates the activities of both the Sec14p/Golgi pathway and the pathway through which transcription of phospholipid biosynthetic genes is derepressed.